P393: SYNERGISTIC EFFECT OF EZH2 INHIBITOR WITH BCL2 INHIBITOR BY ENHANCING PIK3IP1 EXPRESSION IN ACUTE MYELOID LEUKEMIA
Authors: C. Yang, C. Song, Z. Ge
Journal: HemaSphere (2022)

Background: Acute Myeloid Leukemia(AML) is a highly heterogeneous blood cancer with poor outcomes. PI3K Interacting Protein 1(PIK3IP1) binds to the p110 catalytic submit and downregulate PI3K activity. EZH2 is the catalytic subunit of polycomb repressive complex 2(PRC2). Inhibition of EZH2 activity is a therapeutic vulnerability in cancer cells. Combination of the BCL2 inhibitor Venetoclax(Ven) with hypomethylating agents has been approved for treatment in AML with substantial benefits, however, most patients develop resistance. Thus, a more efficacious treatment by targeting the drug resistance paradigm based on BCL2 inhibitor is required. Aims: To investigate the anti-leukemic activity of the combination of the EZH2 inhibitor DZNeP with BCL2 inhibitor Ven and to evaluate the association of PIK3IP1 with clinical characteristics of patients with AML. Methods: EZH2 and PIK3IP1 mRNA level was examined by qPCR in newly-diagnosed AML patients and healthy control from Feb. 1, 2016, to Jan. 30, 2022, at our institute with approval of the Ethics Committee. Cell Counting Kit-8 assay(CCK8) was used for cell proliferation and cytotoxicity in U937 and MV-4-11 AML cells treated with vehicle control, EZH2 inhibitor(DZNeP), Ven, and Combination (Ven+DZNeP). Synergistic effect was analyzed with Calcusyn. RNA-seq was performed with total RNA isolated from U937 cells treated with 2μM DZNeP for 48 hours. Apoptosis was measured by cell staining with Annexin V+propidium iodide (PI) following flow cytometry analysis. Lentiviral PIK3IP1 shRNA knockdown was performed in AML cells. Gene expression was analyzed by qPCR and Western blot. Results: EZH2 mRNA level was significantly increased in 53 newly diagnosed patients with AML compared to 20 healthy bone marrow control(p<0.0001)(Fig.1A). Both DZNeP and Ven had a dose-dependent and time-dependent effect on cell proliferation arrest in AML cells. DZNeP significantly sensitized the effect of Ven on cell proliferation arrest compared to single drug-only control. CalcuSyn analysis showed the synergistic effect of the Ven+DZNeP(Fig.1B). The total apoptosis rate increased significantly in the Ven+DZNeP group(Fig.1C). RNAseq analyses showed that PIK3IP1 is dramatically upregulated(Fig.1D). GSEA enrichment analyses showed that the Differentially Expressed Genes mainly upregulated apoptosis and downregulated cell cycle pathway(Fig.1E). Moreover, the PIK3IP1 mRNA level was significantly decreased in the aforementioned AML cohort compared to the control(P<0.005)(Fig.1F). The AML patients were divided into two PIK3IP1High expression groups(n=29) and PIK3IP1Low expression groups(n=24). PIK3IP1low expression is significantly associated with a lower overall response rate (96.55% vs 79.17%, p=0.047), higher relapse or death rate(58.33% vs 24.14%, p=0.029). A moderately negative correlation between EZH2 and PIK3IP1 was observed(r=-0.376, p=0.006)(Fig.1G). In addition, qPCR data showed that PIK3IP1 is significantly upregulated by DZNeP or Ven+DZNeP(Fig. 1H). PIK3IP1 is efficiently knocked down in U937 and MV-4-11(Fig.1I), and PIK3IP1 knockdown significantly blocked the combination of DZNeP and Ven mediated cell proliferation arrest and apoptosis(Fig.1J-K). Image:Summary/Conclusion: The combination of EZH2 inhibitor DZNeP and BCL2 inhibitor Venetoclax exhibited a strong synergistic cell proliferation arrest and apoptosis of AML cells in PIK3IP1 dependent manner. PIK3IP1low expression is associated with oncogenic markers and poor outcomes in AML. Our results highlight the likelihood of further in vivo pre-clinical study for the potential clinical trial of the novel combination in AML patients.